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Journal of Plant Physiology 163 (2006) 629—637

KEYWORDCell injuryRelative wcontent;Salinity;Na+ toxicitWater defitolerance

0176-1617/$ - sdoi:10.1016/j.

AbbreviationGYR, grain yielrelative water�Correspond

92 41 2651961+92 41 2654213

E-mail addr

www.elsevier.de/jplph

The use of cell membrane stability (CMS) techniqueto screen for salt tolerant wheat varieties

Shafqat Farooq�, Farooqe Azam

Nuclear Institute for Agriculture and Biology (NIAB), P. O. Box. No. 128, Jhang Road, Faisalabad, Pakistan

Received 8 December 2004; accepted 8 June 2005

S;ater

y;ciency

ee front matter & 200jplph.2005.06.006

s: CMS, cell membrand reduction, MSI, memcontenting author. Tel.:(Direct line), 92 41.ess: shafqat_niab@ho

SummaryCell membrane stability (CMS) technique was used to screen salt tolerant (V1, V2),salt sensitive (V5) and two salt/water deficiency tolerant wheat genotypes (V3 and V4)using 100–250mM NaCl salinity maintained in pots containing gravel and nutrientsolution. The objectives were to study: (i) the reliability of CMS technique forscreening wheat under high salinity, (ii) factors that impart stability and/or injury tothe cell membrane, and (iii) the relationship of CMS with other physiologicalparameters affected by the salt stress. Generally, cellular injury increased withincreasing salinity levels. In V5, it was the highest (74.2%) at 250mM, probably due tocombined effect of Na+ toxicity and low (54%) relative water content (RWC). In V1,RWC was similar to that in V5 but injury was comparatively low possibly due to lowconcentration of Na+. The difference between V1 and V2 was significant, either due tothe highest concentration of K+ or the lowest reduction in RWC in V2. In V3 and V4,injury was the lowest at all salinity levels and was within the range of values observedearlier for drought tolerance. A significant negative correlation was detected betweencellular injury and RWC for V1 and V5 but not for V3 and V4. Cellular injury alsoshowed a significant positive correlation with Na+ and a negative correlation with K+

and grain yield (GY). It appeared that CMS technique is suitable for screening wheatunder high salinity levels and for detecting differences that may arise due tocumulative effects of salinity and reduced water contents.& 2005 Elsevier GmbH. All rights reserved.

5 Elsevier GmbH. All rights rese

e stability; GY, grain yield;brane stability index; RWC,

+92 41 2654221 30 (PMBX),2650101 (Residence); fax:

tmail.com (S. Farooq).

Introduction

Salinization of soils has often been considered amajor constraint for agricultural productivity.Nevertheless, the inherent plasticity in some cropplants enables them to grow on such lands asreported for different wheat varieties (Sairam

rved.

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S. Farooq, F. Azam630

et al., 2002; Wang et al., 2003), wild relatives ofwheat (Farooq et al., 1988, 1989) and other crops(Francois and Maas, 1999). For these and similarother studies (Esechie et al., 2002; Gibberd et al.,2002), different screening media such as hydro-ponics (Jung et al., 2004; Knight et al., 2004),gravel culture (Qureshi et al., 1977) and natural orartificially saline fields (Farooq et al., 1995) havebeen used. Information obtained from these ex-periments has provided clear indications on sensi-tivity or resistance of plants at germination,vegetative/generative stages, and at maturity(Reddy and Iyengar, 1999). Because salt toleranceof plants is not simply a tolerance against toxicityof Na+, but also an adaptation to its secondaryeffects like water deficiency/depletion (Munns etal., 2002); the latter has been considered as one ofthe most common and serious consequences ofsalinity (Tabaei-Aghdaei et al., 2000) that results inmalfunctioning of the cellular membranes byincreasing their permeability to ions and electro-lytes. It was therefore considered worthwhile totest a salinity procedure that can collectivelymeasure injurious effects of salts as well as ofwater stress in order to assess real salt tolerancepotential and the factor(s) imparting resistance toa particular plant against these stresses. Measure-ment of cell membrane stability (CMS) is one suchtechnique (Sullivan, 1972) that has often been usedfor screening against drought tolerance in variouscrops such as Sorghum bicolor (Sullivan and Ross,1979), wheat (Blum and Ebrecon, 1981), maize(Premachandra et al., 1989), Populus deltoids(Michael et al., 1994), rice (Tripathy et al., 2000),wheat and wild relatives of wheat (Farooq andAzam, 2002b). Sairam et al. (2002) have usedmembrane stability index (MSI) as one of theparameters to differentiate two wheat genotypesgrowing at salinity levels between electrical con-ductivity (EC) of 5.4 and 10.6 dSm�1 (app.50–100mM). They reported salinity-induced reduc-tion in MSI and relative water content (RWC) in bothgenotypes.

In the present study, we used CMS technique toassess salt tolerance potential of five geneticallydifferent wheat genotypes including salt tolerant,salt sensitive and water deficiency tolerant wheatgenotypes. Salinity levels of 100, 150, 200 and250mM (much higher than those reported earlier bySairam et al. (2002)) were used. The objectiveswere to study: (i) the effectiveness and reliabilityof physiological techniques such as CMS for screen-ing wheat material under high salinity, (ii) thefactor(s) that impart stability and/or injury to thecell membranes at such levels, and (iii) therelationship of CMS with other physiological para-

meters such as RWC, and accumulation of K+ andsugars in the cytoplasm. Since CMS technique isbeing used for assessing salinity tolerance poten-tial, wheat genotypes known for their responses tothe salt stress were utilized.

Material and methods

Wheat material

Material used in this study included a salttolerant wheat variety V1, known to maintain ahigh K+/Na+ ratio under salinity levels rangingbetween EC 6 and 10 dSm�1 (Qureshi, 1985). Thisvariety is also one of the parents of three breedinglines (V2, V3 and V4) produced through crossing V1with a salt tolerant accession of Aegilops cylindrica(Farooq et al., 1992). Among these, V2 can take upmore K+ than Na+ (Farooq et al., 1995) and producea grain yield (GY) higher than V1 under salinitylevels of EC 12–15 dSm�1 or even higher (Farooq etal., 1998). Wheat lines V3 and V4 are also salttolerant, but compared to the commercial culti-vars, require less water and fertilizer to grow(Farooq and Azam, 2002a). A salt sensitive com-mercial wheat variety (V5) was also used. Adetailed description of the materials is providedin Table 1.

Seed germination and plant growth

All genotypes were tested for germination onfilter papers soaked with distilled water containingthe level of 0 (control), 100, 150, 200, and 250mMwith NaCl. Uniform seeds of all the test materialswere placed in Petri plates lined with moist filterpaper. For each genotype, 30 seeds per plate wereplanted with three replications using randomizedcomplete block with split plot design and the datasubjected to analysis of variance (ANOVA). Allplates were kept at 25/15 1C day/night tempera-tures and 60–70% relative humidity. Germinationdata were recorded 5–10 days after planting andone-week-old seedlings were transferred at nineplant pot�1 and three pots genotype�1 in plasticpots (15 in diameter) containing gravel and Hoag-land nutrient solution (Hoagland and Arnon, 1950)salinized to the level of 0 (control), 100, 150, 200and 250mM with NaCl. Three plants in each potwere allowed to grow to maturity and wereharvested in the month of May. Grain yieldcollected from three plants pot�1 were computedand converted into g pot�1. The remaining sixplants in a particular pot were used (two plants

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Table 1. Description of material

Nos.a Variety/Lineb

Pedigree Description Status Reference

V1 LU-26 Khushal/Blue Silver Medium tall, earlymaturing, rustsusceptible

Salt tolerant variety Qureshi et al. (1985)

V2 WL-1073 LU-26/Ae.cylindrciaD//LU-26

Medium tall, earlymaturing,

Salt tolerantbreeding line

Farooq et al. (1992),Farooq et al. (1995,1998)

V3 WL-1076 LU-26/ Ae. cylindricaD//Pak-81

Normal height andmaturity

Salt and waterdeficiency tolerantline

Farooq et al. (1992),Farooq and Azam(2002b)

V4 WL-41 LU-26/ Ae. cylindricaD//Pak-81

High tillring, highyielding

-do- Farooq et al. (1992),Farooq (2000)

V5 Inqbal-91 A selection from Int.CIMMYT nurseries

Medium tall, highyielding

Salt sensitivecultivar

Anonymous (1992)

aWheat material is mentioned according to decreasing order of salinity tolerance (recorded with respect to reduction in grain yield)i.e. V1 most salt tolerant and V5 least salt tolerant (or salt sensitive).bVarieties and cultivars are being used for commercial cultivation whereas breeding lines are under field trials and have not yet beenreleased for commercial cultivation.

Cell membrane stability salt tolerant 631

each) for determining RWC, cellular injury andchemical analyses. The experiments were con-ducted in the months of November–January, whentemperatures range between 15 and 20 and 5 and10 1C for day and night, respectively, with relativehumidity of about 50–55% and day length of o10 h.

Relative water contents (RWC)

For determination of RWC, fresh leaves weredetached from each treatment, replicate, andgenotype and weighed immediately to record freshweight (FW), followed by dipping half of theirportion in distilled water for 12 h. The leaves wereblotted to wipe off excess water, weighed to recordfully turgid weight (TW), and subject to oven dryingat 70 1C for 24 h to record the dry weight (DW). TheRWC were determined by the equation proposes byTurner (1986) that is RWC ¼ [FW�DW]� 100/[TW–DW].

Measurement of cell membrane stability orcellular injury

CMS was determined according to the method ofSullivan (1972). For this purpose, a fully expandedyoung leaf (5th leaf) was selected from eachgenotype, treatment and replication. Twentypieces (1 cm diameter) cut from these leaves weresubmerged into distilled water contained in testtubes. The tubes were kept at 10 1C in a cooledincubator for 24 h, followed by warming at 25 1C

and measuring the electrical conductivity N(C1) ofthe contents. The leaf samples were then killed byautoclaving for 15min and electrical conductivityof the medium measured again (C2). Cellular injurywas determined by using the equation: ½1� ð1�T1=T2Þ=ð1� C1=C2Þ� � 100 proposed by Sullivan(1972) where T and C refer to treatment andcontrol, respectively, and 1 and 2 refer to con-ductivity one and two.

Chemical analysis

For chemical analysis, cell sap extracted bycentrifugation of frozen leaf samples (5th leaf)was analyzed for K+, Na+ (by flame photometer),and total sugars by the enthrone method of Yoshidaet al. (1972).

Results

Seed germination in all of the wheat genotypeswas affected considerably at 100 and 150mM, anddrastically at 200 and 250mM (Fig. 1). Reductionwas more profound in V1 (tolerant variety) and V5(sensitive), in which seed germination reduced to60% and 52% of control, respectively, at 250mM.Compared to V1, seed germination in V2 (tolerantline) was significantly higher and negligibly differ-ent from V3 and V4, the water deficiency tolerantlines (Fig. 1).

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Under control conditions, RWC differed signifi-cantly among different genotypes. It varied be-tween 81% and 84% for salt and water deficiencytolerant (V3 and V4, respectively) and 87% for salttolerant breeding line V2 and 90% for salt tolerant(V1) and sensitive (V5) cultivars (Fig. 2a). Salinity-induced reduction in RWC was also significant, butthe pattern of reduction differed among the salttolerant, salt sensitive, and salt+water deficiencytolerant genotypes. In V1 and V5, RWC decreasedbeyond 30% at 250mM NaCl, whereas in V3 and V4(salt and water deficiency tolerant lines), reductionof this magnitude in RWC was not observed even at250mM NaCl. Breeding line V2, however, exhibitedthe lowest reduction (21% of control) in RWC at thesame salinity level (Table 2).

Cellular injury increased significantly in allgenotypes at all salinity levels, but the magnitudeof increase was more profound in V1 and V5.Between the two, it was higher in V5, especially at

0

15

30

45

60

75

90

105

V1 V2 V3 V4 V5Wheat varieties

Rel

ativ

e w

ater

co

nte

nts

(%

)

1.5 100 150 200 250

Figure 2. Relative water contents (left) and cellular injury (rlevels of 0 (control), 100, 150, 200 and 250mM NaCl.

0

25

50

75

100

125

V1 V2 V3 V4 V5

Varieties

% g

erm

inat

ion

1.5 100 150 200 250

Figure 1. Seed germination in different wheat genotypeson filter paper soaked with water containing 0 (control),100, 150, 200 and 250mM NaCl.

200 and 250mM (61.7% and 74.2%) than in V1 (47.4%and 59.3%), which in turn was significantly higherthan that in V2 (40.7% and 43.1%) at the samesalinity levels. Compared to V1 and V2, injuryremained significantly low in V3 and V4 (salt andwater deficiency tolerant genotypes). In V3, itranged between 26.7% and 31.7% and in V4,between 27.6% and 35.1% at salinity levels of100–250mM NaCl (Fig. 2b).

There was no significant correlation (r ¼ �0:723,�0.750) between RWC and cellular injury in V3 andV4 at any of the salinity levels, but in V1 and V5,RWC and cellular injury were significantly (po0:01)and negatively (�0.900, �0.925, respectively)correlated, especially at 200 and 250mM NaCl.Cellular injury was also significantly (po0:05) andpositively correlated with Na+ (r ¼ 0:910) andnegatively with K+ (r ¼ �0:869) and GY(r ¼ �0:895), which in turn appeared significantly(po0:001) and negatively (r ¼ �0:990) correlatedwith Na+ concentration. Cellular injury and totalsugars also exhibited negative correlation but thevalues were only significant (po0:05) in V3 and V4(r ¼ �0:880 and �0.890, respectively).

Considerable differences were observed for con-centrations of Na+, K+, and total sugars in the cellsap (Table 3). Concentration of Na+ differedsignificantly between control and 100mM NaCl.With further increase in salinity, a progressiveincrease was observed in each genotype. Thelowest Na+ concentrations were observed in V3and V4, and the highest in V5 at all salinity levels.Between V3 and V4, the latter exhibited a higherNa+ concentration, but it was not considerablydifferent from V1 at any of the salinity levels(Table 3).

0

10

20

30

40

50

60

70

80

90

V1 V2 V3 V4 V5

Varieties

Inju

ry %

ag

e

100 150 200 250

ight) in different wheat genotypes as affected by salinity

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Table 3. Concentrations of Na+, K+ and total sugars (mmol L�1) in cell sap of five different wheat genotypes growingunder salinity levels of 0 (control), 100,150, 200 and 250mM NaCl

Variety Salinity levels (mM NaCl)

Control 100 150 200 250

Na+

V1 16.1 b 25.2 b 27.5 b 39.5 ab 45.7 bV2 15.0 ab 29.4 c 29.9 c 41.2 b 44.8 bV3 13.0 a 20.5 a 23.9 a 37.5 a 36.2 aV4 17.4 b 21.8 a 26.6 b 38.9 ab 37.2 aV5 21.7 c 30.3 c 35.0 d 47.7 c 49.5 c

K+

V1 108.5 b 127.8 ab 163.5 d 170.5 c 110.9 bV2 110.0 b 153.0 d 215.5 e 220.3 d 125.5 dV3 101.5 a 125.0 a 131.5 a 145.7 a 103.8 aV4 102.0 a 129.4 b 135.6 b 146.5 a 104.6 aV5 127.2 c 135.0 c 149.9 c 150.3 b 119.9 c

SugarsV1 208.0 b 213.9 b 227.3 c 207.8 b 198.0 bV2 213.0 c 220.2 c 218.3 b 199.0 a 181.3 aV3 246.0 e 237.8 e 235.9 d 223.3 c 230.8 cV4 235.8 d 227.0 d 227.7 c 209.3 b 196.8 bV5 180.0 a 187.0 a 190.7 a 195.5 a 193.8 b

Figures followed by the same letters in one column are not significantly different from each other at 5% level of significance accordingto Duncan’s multiple range test (DMRT).

Table 2. Percent reduction in relative water contents of wheat genotypes under different salinity levels

Wheat genotypes Salinity levels (mM NaCl)

100 150 200 250

V1 (Salt tolerant variety) 9.00 d 13.7 c 25.0 cd 38.2 cV2 (Salt and WD tolerant line) 1.20 b 05.8 a 15.0 a 21.3 aV3 (Salt and WD tolerant line) 1.90 b 06.3 a 19.8 b 24.0 bV4 (Salt and WD tolerant line) 0.07 a 09.2 b 21.1 bc 24.9 bV5 (Salt sensitive cultivar) 7.35 c 22.0 d 23.5 c 39.5 c

Figures followed by the same letter in one column are not significantly at 5% level of significance according to DMRT.WD, waterdeficiency.

Cell membrane stability salt tolerant 633

Significant differences were also observed for K+

concentration. Both V3 and V4 exhibited the lowestK+ concentration under control conditions, whichincreased steadily up to 200mM NaCl, and thendecreased to the lowest (103.8 and104.6mmol L�1, respectively) level at 250mMNaCl. In the remaining genotypes, K+ concentrationalso increased with increasing salinity levels; thehighest level (220.3mmol L�1) observed in V2 at200mM NaCl. Generally, salinity-induced increasein K+ concentration was lower in V3 and V4, andhigher in V2 compared to the remaining genotypes(Table 3).

Under control conditions (0 NaCl), concentra-tions of total sugars were the highest in V3,followed by V4, and decreased with increasingsalinity levels. Nevertheless, it was the highest inboth V3 and V4 at all the salinity levels. In theremaining genotypes, total sugars increased up to150mM NaCl and then decreased with maximumreduction being observed in V2 at 250mM NaCl. InV5, total sugars were the lowest under non-saline(control) conditions, but unlike other genotypes,increased with increasing salinity levels. Never-theless, total sugars remained second lowest (afterV2) in V5 at 250mM NaCl (Table 3).

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Table 4. Absolute grain yield (g pot�1) and reduction over control (%) observed in five genetically diverse wheatgenotypes growing under different salinity levels

Wheat material Salinity levels (mM NaCl)

0 (control) 100 150 200 250

V1 35.9 ab 27.9 b (22.8) 26.7 b (25.6) 16.8 b (53.2) 12.5 ab (65.2)V2 34.8 b 33.5 c (3.7) 31.0 b (10.9) 22.3 c (35.9) 15.0 b (56.9)V3 48.4 c 45.3 d (6.4) 42.8 c (11.6) 33.0 d (31.8) 20.9 c (56.8)V4 59.4 d 48.12 d (19.0) 48.41 d (18.5) 42.7 d (28.11) 19.0 c (68.1)V5 30.2 a 22.2 a (26.5) 17.5 a (40.05) 12.4 a (59.9) 9.8 a (67.5)

Figures in parenthesis are % reduction over control.Figures followed by same letters in one column are not significantly different from each other at 5% level of significance according toDuncan multiple range test (DMRT).

S. Farooq, F. Azam634

GY of all the test materials differed significantlyboth under control and saline conditions. Maximumgrain yield (59.4 g pot�1) was achieved in V4followed by V3 (48.4 g pot�1), while V5 exhibitedthe lowest (31.2 g pot�1) under control conditions.All salinity levels reduced grain yield differentlybeyond 150mM NaCl, except in V5 and V1, wherereduction in grain yield (GYR) was significant at100mM (23 and 26% of control, respectively). In V3and V4, GYR was less than 50% compared to 60% and53% in V5 and V1, respectively, at 200mM NaCl.Between V3 and V4, GYR was higher in V4 than in V3at all salinity levels (Table 4).

Discussion

As mentioned earlier, CMS has often been used toassess drought and salinity tolerance potential ofdifferent crops including wheat (Blum and Ebrecon,1981). Additionally, it has also been used forassessing tolerance to frost (Dexter, 1956), heat(Martineau et al., 1979) and desiccation (Bewley,1979). In most of these studies, CMS exhibited apositive correlation with osmotic potential, K+

concentration, osmotic adjustment, and/or rela-tive water contents: the parameters that areequally affected by salinity stress (Munns, 2002).However, depletion of water has usually beenconsidered as one of the major causes of increasedcell membrane permeability of plants growingunder salt stress (Tabaei-Aghdaei et al., 2000);hence, the first pre-requisite to conduct thepresent study was to select appropriate saltconcentrations that can reduce water availabilityto a level capable of inducing water stress, andsubsequently, the injury to the cell membrane. Weused seed germination as an index for this purpose.Since wheat can tolerate considerably high levels of

salinity at the germination stage (Kingsbury andEpstein, 1984), it was necessary to determinesalinity level(s) that can significantly reduce seedgermination of even the known salt tolerantvarieties. In the present study, these levels turnedout to be 200 and 250mM: the levels that areroughly equivalent to 45–55% of seawater salinity.These levels were selected on the basis of ourprevious experience (Farooq et al., 1994) in whicha salinity level of EC 25 dSm�1 (approximately 50%seawater salinity) reduced seed germination in V1(the salt tolerant variety) by 20% compared tocontrol. A higher salinity level that could reducegermination 420% was therefore selected alongwith the lower levels for comparison.

We observed a progressive increase in cellularinjury and reduction in RWC with increasing salinitylevels. This was consistent with, but significantlyhigher than, the values reported earlier by Sairamet al. (2002). For example, at 250mM NaCl, thesensitive cultivar (V5) exhibited reduction in RWCbeyond 30%, the lowest level after which wheatplants do not survive even if they are re-watered(Blum, 1996). A reduction of this magnitude (39.5%)in RWC usually occurs in plants growing undersevere drought conditions (Tripathy et al., 2000).But under saline environment, reduction up to thislevel is also not surprising, because salinity lowersthe water potential in the roots, and unless thevariety to be tested is drought tolerant, reductionin RWC up to this level would be considered as‘‘water stress’’ for that particular genotype. Undersuch a situation, salinity and water deficiency willboth cause damage to the growing material,including injury to the cell membrane, which inthe present study was more than 74% for V5.

In glycophytes such as wheat, such a high level ofinjury to cell membrane may not occur if thegenotype can avoid toxic levels of NaCl reachingthe leaves or make osmotic adjustment by accu-

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Cell membrane stability salt tolerant 635

mulating higher levels of K+ in the cytoplasm(Sairam et al., 2002) or other organic solutes suchas proline (Hasegawa et al., 2002), glycine betaine(Sakamoto and Murata, 2002), soluble sugars(Maggio et al., 2000), Vitamin B6 (Shi et al.,2002) and through production of antioxidant en-zymes (Hernandez et al., 2000; Sairam et al., 2002)depending upon the genetic constitution of the testmaterial. The salt sensitive (V5) cultivar apparentlylacks the ability to keep Na+ away from thecytoplasm and thus absorbed high amounts(21.7–49.5mmol L�1) of Na+ that fall in the ranges(25–45mmol L�1) reported earlier for the sensitivewheat cultivars (Iqbal et al., 2001), durum wheatvarieties and synthetic hexaploids (Gorham,1990a). This high amount of Na+ further increasedwhen a drastic reduction in RWC occurred underthe influence of various salinity levels. Thus,cumulative effects of salt and reduction in RWCcaused the highest level (74%) of cellular injury inV5, which appeared significantly and negativelycorrelated with RWC. Salt tolerant variety V1,however, absorbed less Na+ compared to V5, butreduction in RWC to 39% probably caused in V1cellular injury of the order of 59% at the samesalinity level. It appeared that V1, which cantolerate salinity up to EC 10dSm�1 (Qureshi,1985), is sensitive to reduced water contents thatwere significantly low beyond 150mM NaCl. In thepresent study, such reduction in RWC has beenconsidered as water deficiency that might also haveresulted into GYR of 53% and 65% (highly unecono-mical) in V1 at 150 and 200mM NaCl, respectively.

Compared to V1 (salt tolerant variety), V2 (salttolerant breeding line) absorbed higher amounts ofNa+ but showed significantly lower cellular injurycompared to V1. Probably the lowest reduction inRWC and the highest levels of potassium helpedkeep the injury low. Potassium-mediated osmoticadjustment has been reported in drought tolerancein barley (Blum, 1989) and has also been provedeffective in freezing and salinity tolerance as bothinvolve a component of water stress (Blum, 1988).

The lowest cellular injuries were observed in V3and V4. These lines absorbed the lowest Na+

(20–37mmol L�1), possibly due to the fact thatthese lines have genes transferred from Ae.cylindrica (Farooq et al., 1992) that possessedone (DD) of the two (CCDD) genomes similar to thatof Ae. tauschii (Ae. squarrosa), which is known tohave low leaf Na+ and high K+ concentrations and ahigh K+/Na+ ratio (Gorham, 1990b). The Ae.cylindrica accession used in the present study waslater found to be drought tolerant (Farooq andAzam, 2002b). It was probably due to this factorthat both V3 and V4 exhibited the lowest RWC

under control (non-saline) as well as saline condi-tions. Such low RWC were apparently adjusted byaccumulating sugars that were the maximum, andsignificantly higher in V3 compared to V4. Althoughsugars are known for their osmotic influences inwheat (Blum and Ebrecon, 1981) and maize (Pre-machandra et al., 1989) growing under drought aswell as salinity and in P. deltoides growing underdrought conditions (Michael et al., 1994), itsaccumulation to the level observed in V3 and V4has never been reported . Such high accumulationof sugars should have been at the expense of yieldas this is an energy-consuming process (Raven,1985). Nevertheless, the yield was not reducedsignificantly both in V3 and V4 even at 200mMNaCl. This would suggest that compared to salinitythese lines are probably more tolerant to waterdeficiency, which might have been transferred fromAe. cylincrica. It can be inferred, therefore, thatboth the lines are primarily tolerant to waterdeficiency and then to salt and confirms the earlierfindings of Farooq and Azam (2002b) that salt anddrought tolerance co-exists. This observation wasfurther strengthened when we compared the valuesof cellular injury observed in the present study withthose reported previously (Blum and Ebrecon,1981) for wheat and barley as a result of desicca-tion induced by PEG and not due to salinity per se.The variations observed for injury in the Blum’sstudy ranged between 11.2% and 31.4% for droughtresistant and between 46.9% and 79.6% for suscep-tible varieties of wheat. We observed variation forcellular injury that ranged between 42.2 and 74.2for sensitive cultivar (V5), 38.2 and 59.3 fortolerant variety (V1), and 36.4 and 43.1 for tolerantline (V2) but for water deficiency tolerant breedinglines (V3 and V4) these values ranged between 26.7and 31.8 and 27.6 and 35.1, respectively. Based onthese variations, V3 and V4 appeared more salttolerant than the salt tolerant variety (V1) andbreeding line (V2). This has again confirmed ourearlier observation (Farooq and Azam, 2002b) andalso of Munns (2002) that salt tolerant plants dopossess some degree of tolerance for waterdeficiency, and that the plants without this abilitymay not appear more salt tolerant than those whichdo possess this ability. The salt tolerant variety (V1)acts as an example of this.

Conclusion

Salt tolerance is usually associated with com-parative biomass (or grain yield) reduction whenplants are exposed for longer periods of time to a

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S. Farooq, F. Azam636

saline environment. Since effect of salinity at everystage of plant growth is different (Munns et al.,2002), physiological parameters apart from the K+/Na+ ratio have not so frequently been used for salttolerance screening. Compared to this, GYR hasusually been taken as an index of tolerance,despite the fact that screening of germplasm basedon GYR is not feasible, particularly for largequantities as it takes longer time. Even with theK+/Na+ ratio, it is difficult to assess whether lowleaf Na+ is due to ion selectivity that allows theplants to discriminate against Na+ (salt toleranceonly), or whether it is because of water deficiencytolerance or low water requirements of the plant,or both. With physiological parameters such asCMS, differences in the tolerance for compoundstresses such as salinity and water deficiency can bedetected, which is an added advantage. In addi-tion, differences between closely related speciescan also be detected, which are not possible withGYR or K+/Na+ discrimination, despite the fact thatthe former is significantly and negatively correlatedwith GY. Consequently, CMS can be used moreeffectively than GY/GYR for screening large quan-tities of germplasm at seedling stage.

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